Previous work in this project demonstrated that extracts of sediments from an estuary adjacent to a PAH-dominated Superfund site in VA are highly teratogenic to embryos of the killifish, Fundulus heteroclitus, with cardiovascular defects the most pronounced effect observed. Studies with model PAHs demonstrated marked synergistic interactions between PAH-type AHR agonists and CYP1A inhibitors. Current risk assessment approaches for PAHs, however, assume an additive model of toxicity for PAHs. Hypoxia is an important variable affecting development as well as comprising a natural stressor in estuaries. Thus, the overarching goal of this project is to elucidate interactive effects on development and mechanisms underling such effects by PAH mixtures and hypoxia in Fundulus. The Specific Aims are: 1. To characterize the effects of a Superfund site PAH mixture on development in Fundulus with imaging, biochemical and molecular tools. 2. To elucidate interactive effects and underlying mechanisms of representative CYP1A inducers (AHR agonists) and inhibitors on development. 3. To elucidate interactive effects and underlying mechanisms of model PAHs and hypoxia on development in Fundulus. 4. To determine effects of selected PAH mixtures on development in other models employed in the Center. 5. To develop practical biomarkers for developmental effects of hydrocarbon mixtures in vertebrates. This study will integrate three complimentary experimental methodologies: (1) imaging and pathology, (2) analysis of gene expression, particularly by microarray, and (3) biochemical analyses, particularly of responses mediated by the aryl hydrocarbon receptor pathway, the hypoxia inducible factor pathway, and oxidative stress. This project will elucidate toxic interactions of a class of chemicals (PAHs) that is exhibiting steady increase in the environment, invariably occurs in mixtures, and for which current assessments may underestimate risks for developmental effects. The model for this project, Fundulus, has great ecological relevance, is an excellent sentinel species for coastal ecosystems, and provides a useful model for developmental studies relevant to human health.